Our group has identified flavivirus non-structural protein-1 (NS1) as a key immune evasion protein that antagonizes the complement system. We have identified a novel mechanism by which the 50 kDa secreted NS1 interacts with factor H, inhibits C4 activation and enhances C4b degradation. In mice, levels of C4 decrease soon after WNV infection, correlating with a spike in NS1 antigenemia. As both C4-/- and C4+/- show increased susceptibility to lethal WNV infection, we hypothesize that low levels of C4 in humans may be a risk factor for severe flavivirus disease. In humans, complement activation (low C4) immediately precedes the onset of dengue hemorrhagic shock and correlates with the development of this syndrome. Further, the mechanism whereby NS1 protein binds selectively to human cells is based on their GAG profile. The goal of this highly collaborative and interactive grant proposal is to identify the structural and molecular basis of the inhibitory actions of flavivirus NS1 relative to the complement system, and to evaluate whether humans with specific gene copy number variation of C4 alleles are more susceptible to severe flavivirus infection. The long term aim, therefore, is to define the role of NS1 in the immunopathogenesis of flavivirus infections. We will characterize the complement inhibitory function of flavivirus NS1 proteins, identify the structural basis of NS1 function and determine whether C4 copy gene variation is associated with severe flavivirus infection in humans. This grant brings together three seasoned immunologists with special expertise in complement, structural biology and flavivirus pathogenesis. It also features international collaborations with flavivirus groups in Canada, Thailand and Nicaragua. Lastly, it will provide both novel insights into the basic immunobiology evasion strategy of flaviviruses as well as relate these advances to clinical disease states. An enhanced understanding of NS1 biology and structure may foster the development of novel antibodies or small molecule inhibitors that block immune antagonism, increase our understanding of risk, and identify target populations for vaccines or other types of prophylaxis.